With > 1.4 million people dying every year from Mycobacterium tuberculosis (Mtb) infection, tuberculosis (TB) is considered a pandemic, which is increasingly difficult to control. The deadly synergy between TB and HIV infection, and the alarming number of drug-resistant Mtb strains threaten our ability to control TB, even in developed countries. To halt and reverse this development, novel therapeutics that dramatically shorten and simplify TB treatment and that use new drug targets are needed. We recently discovered copper homeostasis as an important virulence factor of Mtb. We can demonstrate that genetic perturbation of Mtb copper homeostasis induces copper hypersensitivity which is associated with a strong virulence defect. Copper ions are employed by macrophages to kill Mtb hence compounds that synergize with copper dependent innate immune functions create novel opportunities for therapeutic interference. In this project we will develop a robust combinatorial high-throughput screening (HTS) assay for the identification of copper-boosting drugs that either (i) inhibit individual components of (ii) or increase copper uptake and thereby overwhelm the intrinsic copper resistance pathways of Mtb. In Aim 1, we will optimize and transfer an HTS screening assay for copper-boosting drugs to a robotic platform utilizing a BSL2 approved mutant of Mtb H37Rv. In Aim 2, we will develop and validate novel and more rapid secondary assays, which will guide hit prioritization. In particular, we will validate and utilize a flow cytometry-based approach to evaluate viability of drug exposed avirulent Mtb cells. This assessment is 1,000 times faster than traditional CFU counts, is HTS compatible, supports kinetic read-outs and requires less than 1,000 individual cells per read and condition. The various assays are designed to test whether the identified hits act as bactericidal or bacteriostatic compounds, exhibit activity against latent (non-growing) Mtb or interact with antiretroviral drugs. In research leading to this application, we have already identified two proof of concept compounds that target Mtb in a copper dependent manner and exhibit an in vitro therapeutic index of >20. In Aim 3, we will perform a pilot screen to evaluate overall assay performance. Upon completion of this pilot campaign, we intend to apply for Fast Track entry of our screening assay into the NIH Molecular Libraries Probe Production Centers Network (Aim 4).